This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are using electron tomography of mouse mammary glands and liver, stained with malachite green, to study the biogenesis of cytoplasmic lipid droplets and lipid rafts. Malachite Green (4-[(4-dimethylaminophenyl)-phenyl-methyl]-N,N-dimethyl-aniline) is an osmiophilic stain that binds to lipids in most cells and tissues. Preliminary EM and tomographic studies suggest that malachite green stains specific areas of phospholipid monolayers and bilayers that seem to correspond with high concentrations of cholesterol in chemically-fixed rat and mouse liver and mammary epithelia. Patch-like staining is observed in the plasma membrane and domains within Golgi cisternae. We hypothesize that the plasma membrane and Golgi domains are mature lipid rafts and nascent lipid rafts, respectively. Dense staining is also observed within cytoplasmic lipid droplets (CLDs) at regions where ER is juxtaposed to the CLD surface. We are working to optimize our preparations by introducing malachite green stain to freeze-substitution protocols so that the method can be used with rapidly-frozen cells and tissues. This approach will allow for high-resolution investigations of cholesterol movement through the cell, as well as the 3D architecture of lipid rafts within the Golgi and at the plasma membrane. Additionally, we will apply this technology to the investigation of cholesterol-dense localizations in cytoplasmic lipid droplets in 3 cell types known to produce CLDs (hepatocytes, mammary epithelial cells, and adipocytes). This staining method, coupled with rapid-freezing, freeze-substitution and EM tomography, will facilitate the understanding of the biogenesis of CLDs and answer the following questions: 1) Are localized areas of concentrated cholesterol juxtaposed to specific cellular membranes? If so, what are they? 2) Do these localized areas correlate with any time point during the biogenesis of the CLDs?